Images acquired on the Nikon N-SIM-S system were reconstructed using stack reconstruction in the NIS elements software. Where stated, post-expansion image data was deconvolved using Huygens professional version 19.04 (Scientific Volume Imaging, The Netherlands; http://svi.nl ). A theoretical point spread function (PSF) was generated based on the microscope parameters, and images were deconvolved using a classical maximum likelihood estimation (CMLE), a non-linear iterative restoration method that optimises the likelihood the objects in the estimated image are correctly localised based on the image and the PSF. This restoration method relies on the generation of an estimate of an object (synthetic image), which is compared to the measured image. The result of this is used to improve the original until the ‘difference’ between the synthetic and measured image reach a minimum. Parameters for deconvolution were tested on example data sets for each experiment to determine optimal values, and these deconvolution templates were used for subsequent image processing and experimental repeats.
N sim s system
Manufactured by Nikon
The N-SIM-S system is a structured illumination microscope designed for super-resolution imaging. It provides high-resolution imaging capabilities by utilizing structured illumination techniques to enhance the spatial resolution beyond the diffraction limit of conventional optical microscopy.
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3 protocols using n sim s system
1
Nikon N-SIM-S System Image Reconstruction
2
Structured Illumination and Confocal Microscopy
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For structured illuminated microscopy (SIM), prepared samples were imaged on the Nikon N-SIM-S system which is combined to Nikon Ti2-E microscope with 100× magnification, 1.49 numerical aperture (NA), 1.515 refractive index TIRF objective lens. The PSF width was 0.129 μm. We used TI2-FL N-SIM 405/488/561/640 Quad Band DM filter cube. Images were captured by 2048 × 2048 pixels using Hamamatsu ORCA-Flash 4.0 digital CMOS camera and reconstructed by using Nikon NIS Element version 5.01. SIM images were taken by 93 steps from 10.96 μm total thickness.
For confocal microscopy, images were acquired using a Nikon A1R confocal microscope with 60× magnification lens (CFI Plan Apochromat VC 60× Oil, NA 1.40). The PSF width was 1.00 μm and the pinhole was set to 1.0 airy unit.
For confocal microscopy, images were acquired using a Nikon A1R confocal microscope with 60× magnification lens (CFI Plan Apochromat VC 60× Oil, NA 1.40). The PSF width was 1.00 μm and the pinhole was set to 1.0 airy unit.
3
Nikon N-SIM-S Deconvolution Imaging Protocol
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Images acquired on the Nikon N-SIM-S system were reconstructed using stack reconstruction in the NIS elements software. Where stated, post-expansion image data was deconvolved using Huygens professional version 19.04 (Scientific Volume imaging, the Netherlands, http://svi.nl). A theoretical point spread function (PSF) was generated based on the microscope parameters and images were deconvolved using a classical maximum likelihood estimation (CMLE), a non-linear iterative restoration method which optimises the likelihood the objects in the estimated image are correctly localised based on the image and the PSF. This restoration method relies on the generation of an estimate of an object (synthetic image) which is compared to the measured image. The result of this is used to improve the original until the "difference" between the synthetic and measured image reach a minimum. Parameters for deconvolution were tested on example data sets for each experiment to determine optimal values, and these deconvolution templates were used for subsequent image processing and experimental repeats.
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